All types of cells sense and respond to stressful environmental factors in order to minimize harm to the cell. We propose to investigate a novel pathway by which bacteria avoid lethal consequences of envelope stress by removing toxic stressors from the cell using vesicles. Outer membrane vesicles offer a means for bacteria to discard toxic insoluble envelope material. Vesicles contain a subset of outer membrane and periplasmic components. They are produced constitutively and without concomitant bacterial lysis. Vesicles have been most recently characterized for their roles in trafficking of virulence factors and immunomodulatory molecules between bacterial pathogens and eukaryotic cells. Overcoming lethal stressors is essential for cellular viability, but it should also be considered that when the stress response promotes secretion of cellular products, it significantly impacts the immediate and distal environment. The overall hypothesis of this study is that Gram-negative bacterial envelope stressors trigger outer membrane vesicle production, thereby modulating virulence properties of pathogens. Little is understood about the molecular and genetic basis regulating vesicle production. Our preliminary results demonstrate specific stress-induced vesicle production triggered by a variety of envelope factors. The proposed experimental approach will be to characterize the molecular triggers of vesiculation by E. coli, to characterize the envelope changes associated with stress-induced vesicle production, and to identify the genetic basis for stress- related vesicle production. This research will impact on human health because the results will yield transformative insight into the understanding of this common bacterial pathogen process associated with virulence factor transmission and inflammation, and potentially uncover new bacterial vulnerabilities that can be exploited with pharmaceutical treatment. PUBLIC HEALTH RELEVANCE: The generation of outer membrane vesicles impacts bacterial survival and allows the dissemination of inflammatory and virulence factors in the stressful host environment. This research plan is designed to transform the general understanding of this bacterial response to stressors. Understanding the mechanics of the vesiculation response through these studies will enable us to help prevent pathogenic bacteria from causing human disease worldwide.